Integrin β1, PDGFRβ, and type II collagen are essential for meniscus regeneration by synovial mesenchymal stem cells in rats

Synovial mesenchymal stem cells (MSCs) injected into the knee promote meniscus regeneration in several animal models; however, the mode of action is unknown. Our purpose was to identify the molecules responsible for this meniscus regeneration. Rat synovial MSCs were treated with neutralizing antibodies for integrin β1, PDGFRβ, or CD44 or with the CRISPR/Cas9 system to delete Vcam1, Tnfr1, or Col2a1 genes. After partial meniscectomy, rat knees were injected with MSCs, and the regenerated meniscus area was quantified three weeks later. The in vivo and in vitro functions were compared between the treated and control MSCs. Anti-integrin β1 neutralizing antibody inhibited in vitro MSC adhesion to collagen-coated chambers, anti-PDGFRβ neutralizing antibody inhibited proliferation in culture dishes, and Col2a1 deletion inhibited in vitro chondrogenesis. In vivo, the regenerated meniscus area was significantly smaller after injection of MSCs treated with integrin β1 and PDGFRβ neutralizing antibodies or lacking type II collagen gene than after control MSC injection. By contrast, the regenerated areas were similar after injection of control, CD44-, Vcam1-, or Tnfr1 treated MSCs (n = 12–16) MSCs. Synovial MSCs injected into the knee joint promoted meniscus regeneration by adhesion to integrin β1 in the meniscectomized region, proliferation by PDGFRβ, and cartilage matrix production from type II collagen.


Results
Effect of integrin β1 on adhesion and meniscus regeneration in synovial MSCs. Treatment of MSCs with integrin β1 neutralizing antibody (Fig. 1A) significantly inhibited the adhesion of MSCs to collagencoated chambers (Fig. 1B). Three weeks after the meniscectomy (Fig. 1C), the injection of only vehicles (W/o MSCs) resulted in a slight increase in the size of the meniscus due to a natural ability to heal (Fig. 1D, Fig. S1). Injection of IgG-treated MSCs significantly increased the size of the meniscus (Fig. 1E), whereas injection of integrin β1-treated MSCs significantly reduced the effect of MSCs on meniscus size. The regions of meniscus regeneration were histologically comparable in all three groups (Fig. 1D, Fig. S2).
In vivo experiments of meniscus regeneration were also performed by manipulating CD44 and Vcam1. Meniscus regeneration was equivalent for MSCs treated with CD44 neutralizing antibody or with IgG (Fig. S3). The effect of Vcam1 gene knockout was comparable to that of wild-type MSCs (Fig. S4).
Effect of PDGFRβ on proliferation and meniscus regeneration in synovial MSCs. The ATP assay for MSC proliferation at 6 days ( Fig. 2A) showed significantly enhanced proliferation for PDGF-BB (Fig. 2B). Treatment with IgG significantly inhibited proliferation, and anti-PDGFRβ neutralizing antibody treatment further decreased it. Injection of IgG-treated MSCs (Fig. 2C) significantly increased the size of the meniscus (Fig. 2D,E, Fig. S5), whereas injection of PDGFRβ-treated MSCs significantly reduced the effect of MSCs on meniscus size. All three groups showed histological similarities in the regenerated areas of the meniscus (Fig. 2D,  Fig. S6).
In vivo experiments of meniscus regeneration using gene-knockout MSCs were also performed for Tnfr1. MSCs lacking this gene showed equivalent regeneration of the meniscus to that of the wild-type MSCs (Fig. S7).

Effect of type II collagen on cartilage matrix synthesis and meniscus regeneration in synovial
MSCs. The results of the in vitro chondrogenesis assay carried out for 21 days (Fig. 3A) showed an inhibition of cartilage matrix synthesis, including type II collagen expression, in Col2a1-knockout MSCs (Fig. 3B). The cell pellet was significantly smaller and lighter in Col2a1 knockout MSCs than in Col2a1 wild-type MSCs (Fig. 3C,D ). The in vivo experiment for meniscus regeneration (Fig. 3E) also showed a significantly smaller regenerated meniscus area following treatment with Col2a1 knockout MSCs than with Col2a1 wild-type MSCs, but this area was significantly larger following treatment with Col2a1 knockout MSCs than with vehicle-treated MSCs (W/o MSCs) (Fig. 3F,G , Fig. S8). No obvious histological difference was detected in the regenerated area of the meniscus in the three groups (Fig. 3D, Fig. S9).

Discussion
Transplantation of synovial MSCs has been shown to promote meniscus regeneration 11,[13][14][15][16][17][18] , but the molecules and signals that are essential for this process remain obscure. Previous regeneration studies of MSCs injected into tissues other than meniscus and articular cartilage have identified important molecules and signals, including integrin β1 20,27 , VCAM1 21 , and CD44 22 for adhesion, PDGF 23 and TNFα 24 for proliferation, and type II collagen 19,25 for matrix synthesis. In this study, we investigated whether these molecules are essential for the promotion of meniscus regeneration by synovial MSCs, both in vitro and in vivo.
Integrins are representative molecules associated with cell adhesion and with a superfamily of cell adhesion receptors 27 that form αβ heterodimers and adhere to various extracellular matrices 27 . Among them, integrin β1 forms heterodimers with most integrin α subunits and plays a role in adhesion to various extracellular matrices 27 . Our findings showed that inhibition of integrin β1 by a neutralizing antibody inhibited the adhesion of synovial MSCs to collagen-coated chambers and impaired meniscus regeneration after transplantation of synovial MSCs. Horie et al. reported that synovial MSCs injected into the knees of rats with a partially resected meniscus adhered to the dissected edge of the meniscus 15 . These findings suggest that inhibition of integrin β1 prevents meniscal regeneration by interfering with the adhesion of synovial MSCs to the meniscus lesion.
CD44 and VCAM1 are also molecules involved in cell adhesion 21,22 , but neither anti-CD44 neutralizing antibody treatment nor Vcam1 gene knockout decreased the ability of MSCs to regenerate the meniscus. MSCs also express other adhesion molecules, including CD168 28 , ICAM1 21 , and ALCAM 21 , which have similar functions to CD44 and VCAM1 for substrate adhesion. We speculate that these molecules may compensate for the adhesive function of CD44 or VCAM1 when they are absent in synovial MSCs.
PDGF promotes the proliferation of synovial MSCs in cell culture systems 23 . Receptors for PDGF form homodimers or heterodimers, and since synovial MSCs express PDGFRβ most strongly among the receptors 29 , we focused on PDGFRβ. Treatment of PDGFRβ with neutralizing antibodies inhibited PDGF-induced cell proliferation in vitro, and injection of cells treated with PDGFRβ-neutralizing antibodies prevented meniscus regeneration in vivo. Horie et al. reported that intra-articular injection of luciferase-expressing synovial MSCs into rats after meniscectomy increased the luminescence intensity approximately threefold after 3 days 15 . These results suggest that inhibition of PDGFRβ function inhibits the proliferation of synovial MSCs attached to the injured meniscus, resulting in impairment of meniscus regeneration.
TNFα also promotes proliferation of synovial MSCs in a cell culture system 24 . The cellular response to TNFα is induced via two receptors, TNFR1 and TNFR2. TNFR1 is expressed in most tissues, whereas TNFR2 is mainly localized on immune cells 30 ; therefore, we focused on TNFR1. Synovial MSCs lacking TNFR1 did not affect meniscus regeneration in vivo, indicating that TNFα signaling was not essential for the effects of synovial MSCs on meniscus regeneration.
Collagen, which comprises about 20% of the meniscus, is the second most abundant component after water 31 . Col2a1 is a representative gene that encodes type II collagen in the meniscus 32  www.nature.com/scientificreports/  www.nature.com/scientificreports/ blue in vitro, and the produced cartilage pellets were smaller in size for the Col2a1 knockout MSCs than for the controls. In vivo studies demonstrated that Col2a1 knockout in synovial MSCs suppressed meniscus regeneration. Decreased expression of type II collagen due to mutation in Col2a1 has been reported in patients with genetic disorders of cartilage 33 . Chen et al. also reported that the addition of type II collagen protein promoted the production of cartilage matrix in MSCs 34 . The association between type II collagen expression and meniscus formation remains to be fully elucidated; however, these findings show that the Col2a1 gene is essential for the chondrogenic differentiation of synovial MSCs in vitro and for meniscus regeneration in vivo.
We have identified essential molecules in synovial MSCs for meniscus regeneration. Generally, the mode of action is less clear for MSCs administered systemically or intraarticularly than for small molecular compounds and antibody drugs, and items for assessing the quality of cell therapy products are often restricted to viability, www.nature.com/scientificreports/ surface antigens, and in vitro differentiation potential. The molecules identified as essential in this study can serve as quality items and markers for elucidating the efficacy of synovial MSCs destined for use in meniscus regeneration therapy. Approximately 4 elapsed from the time the cells were prepared to the time the cells were injected, and the cells were kept on ice throughout this interval. This may have affected cell viability and activity. However, the cells remaining after completion of the injections to all rats did not show any decreases in viability. Furthermore, no association was noted between the order of the injection and the regenerated meniscus area in rats. These observations indicate that storage of the cells on ice for 4 h did not significantly affect cell viability or activity.
The regenerated meniscus area was used as a measure of the effect of the injected cells. A similar approach has been reported in studies using rats 15 , rabbits 16 , and pigs 17,35 . In those studies, the area of the regenerated meniscus correlated with the stainability with safranin O and type 2 collagen. It also correlated with the degree of inhibition of degeneration of the articular cartilage adjacent to the meniscus. Furthermore, in pigs, it was correlated with the tensile strength to failure of the meniscus 35 . The area of the regenerated meniscus area is a reliable indicator for determine the effect of the injected cells.
This study had several limitations. One was that the in vitro analysis for integrin β1 and PDGFRβ does not reflect the in vivo biological events, so in vivo analyses of adhesion and cell proliferation are still required. Another limitation was that the regenerated meniscus was not quantitatively evaluated in histological tissue sections. However, none of the histological images shown in the Supplementary Figures show any marked differences among the groups. A third limitation was that we did not conduct histological examinations for molecules that did not cause any obvious size differences in the regenerated meniscus area. Had we conducted these examinations, we might have detected some differences. A fourth limitation was that no validation assays were performed for the anti-CD44 antibody, so, the problem of neutralizing action could not be completely ruled out. However, we used antibodies whose blocking action was guaranteed by the manufacturer. A further limitation was that we did not analyze where the injected MSCs would be engrafted in the joint or how long they would remain there. In our previous study using the same rat model, the injected cells accumulated at the site of meniscectomy after 1 day and were still detectable in the regenerating meniscus after 12 weeks, and were expressing type 2 collagen 15 . A last limitation was that we used rat cells and examined the MSCs in rat knees. Although rat and human MSCs have many properties in common, further study is needed to confirm that the results in humans will be similar to those obtained in rats.
The mechanism by which intra-articular injection of synovial MSCs promotes meniscus regeneration can be divided into three steps: adhesion of the MSCs to the area around the meniscus defect, MSC proliferation in the joint, and MSC production of cartilage matrix. In this study, we identified essential molecules for each of these steps. Synovial MSCs injected into the knee joint adhered to the area around the meniscectomized region via integrin β1, proliferated via PDGFRβ, produced cartilage matrix through type II collagen, and promoted meniscus regeneration (Fig. 4). Conversely, CD44, VCAM1, and TNFR1, which were molecules predicted to be important at the start of this study, were deemed nonessential for meniscus regeneration.

Methods
Animals. All animal care and experiments were conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and the institutional guidelines of the Animal Committee that includes the first author as a member. Male/female Lewis rats (6-10 weeks old) were purchased from The Jackson Laboratory Japan, Inc. (Yokohama, Japan) (total of 130 animals). All rats were freely allowed access to food, water, and activity. The rats were maintained under a 12 h dark-light cycle at controlled temperature (20-26 °C) and humidity (30-70%).   (Table S1) were added and the plates were incubated for 2 days. Target gene editing was confirmed by extracting DNA from 1 × 10 5 cells, amplifying the target gene by PCR, and detecting the mutant gene using the GenArt Genomic Cleavage Detection Kit (Thermo Fisher Scientific). For cell cloning, the cells with mutant genes were collected, plated in 96-well plates at a concentration of 0.5 cells/well, and cultured to provide over 10 8 cells for transplantation. To confirm gene knockout, DNA was extracted from 1 × 10 5 cloned cells, and its sequences were confirmed by Sanger sequencing (Fig. S10, Table S2-S4).
Both the right and left knee joints underwent surgery. A medial parapatellar incision and lateral dislocation of the patellar tendon were conducted to expose the medial meniscus. The anterior insertional ligament of the medial meniscus was transected to dislocate the medial meniscus anteriorly, and the medial meniscus was resected at the level of the medial collateral ligament. The capsule was closed using nylon sutures, and MSCs at 5 × 10 6 cells/50 µL or vehicle were injected into the knee joint using a 28G needle. (All MSCs and vehicle were stored on ice until administration). The knee joint was moved three times, and the skin was sutured 15 . Surgical treatment and administration were performed together per condition instead of randomly, in order of cell preparation. The time required was approximately 40 min per animal for both knees. The rats were allowed to walk freely in their cages after the surgery. Eight rats per condition were used for Integrin β1 and PDGFRβ experiments, 6  www.nature.com/scientificreports/ buffered formalin solution and decalcified with 0.5% EDTA (pH 7.5) for 3 days at 4 °C, followed by gradient replacement with 20% sucrose for 24 h at 4 °C. The center of the regenerated area of the meniscus was radially sectioned and histologically observed with safranin-O staining.

Statistical analysis.
Comparisons between the two groups were performed using an unpaired t-test, and comparisons among the three or more groups were performed using one-way ANOVA with Bonferroni's Multiple Comparison Test. Statistical tests were conducted using GraphPad Prism ver. 5.04 (GraphPad Software Inc., San Diego, CA, USA). A P value of < 0.05 was considered statistically significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
Ethical approval. All

Data availability
The data sets obtained and analyzed in the current study are available from the corresponding author on reasonable request.